Rapid and effective protocol to measure glutathione peroxidase activity

Abstract

Background Intracellular hydroperoxides are harmful reactive oxygen species that can cause damage. Cells depend on antioxidant enzymes such as glutathione peroxidase (GPx) to prevent their accumulation. GPx is present in all cell compartments, including the cytosol, mitochondria, and peroxisomes. This article will present a rapid and reliable method for measuring GPx activity, which has been rigorously tested and proven dependable. Results The enzyme samples are incubated in a phosphate buffer containing specific concentrations of glutathione (GSH) and peroxide and then incubated for a suitable time to react before Ellman's reagent (5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB) is added. This method, designed for ease of use, is effective and straightforward. When GSH reacts with DTNB, mixed disulfides (GS-S-DTNB) are formed, where the thiol group replaces one of the nitrobenzoic acid moieties in DTNB. The release of the nitrobenzoic acid group as nitro-2-thiobenzoate anion (TNB−) creates a yellow anion that can be measured spectrophotometrically at a wavelength of 412 nm. The intensity of the yellow color is directly proportional to the unreacted glutathione concentration. GPx activity was linked to decreased absorbance of the TNB− anion, providing a sensitive and relatively specific method for detecting thiol groups in various biological samples. Conclusion This study utilized a Box–Behnken design (BBD) to apply to response surface modeling (RSM) to determine the best conditions for measuring GPx activity. The efficiency of the new protocol was validated by comparing GPx activity in matched samples using the Bland–Altman plot analysis and reference methods. The new protocol was just as accurate as the reference one, with a correlation coefficient of 0.9991. Graphical abstract